This proposal will enable the investigators to participate with scientists in Kenya on a research program aimed at the discovery of regulatory mechanisms that serve to coordinate metamorphosis behavior in tsetse, the vector of African trypanosomiasis. The goal of the initial funding period was to obtain a good overview of the behavior associated with tsetse metamorphosis, but we now focus primarily on a sub-set of these behaviors, parturition and pupariation, two events that appear to be particularly vulnerable to disruption. Tsetse has a peculiar reproductive strategy in which larval development is completed within the uterus of the female, and she gives birth to a fully developed third instar larva that pupariates within an hour after being deposited. Parturition is achieved through a sequence of internal contractions that can be monitored with a sensitive barographic technique. This technique, along with in vitro preparations of uterine muscle, will be used to evaluate the efficacy of brain extracts, known hormones and other agents in eliciting or blocking the parturition response, and at the conclusion of these experiments we anticipate presenting a model explaining how parturition is mediated and how it may be disrupted. In tsetse larvae, unlike other flies, the CNS plays a major role in regulating the onset of pupariation and cuticular tanning. The CNS is the recipient of sensory information that evaluates the suitability of a potential pupariation site, and it is the CNS, that relays this information directly to the epidermal cells through nervous conduits. The site of sensory input will be determined by a combination of selective destruction of small regions of the cuticle and an anatomical examination of cuticular ultrastructure and innervation. Innervation of the epidermis will be examined to understand how the message from the CNS is relayed to the epidermal cells, and this path of transmission will be probed using pharmacological agents and known neurotransmitters. In addition, a series of experiments already underway will determine the environmental cues used to drive the daily rhythm of adult eclosion that peaks in mid-afternoon. This study should provide a basis for understanding metamorphosis behavior and its regulation in tsetse and may thus augment strategies for the control of this important pest species.